We have previously demonstrated the presence of a myosin I like protein in the nucleus. This protein has an apparent molecular weight of 120,000, it is associated with K+-EDTA ATPase activity, it binds calmodulin, it is photoaffinity labelled with ATP, it binds actin in the absence, but not the presence, of ATP and it is recognized by an antibody to myosin I purified from adrenal glands. Microsequencing of the 120 kDa protein has positively identified it as a member of the myosin I beta subfamily of the myosin superfamily of actin-based molecular motors. We have used the microsequencing data to clone the cDNA for this protein. The microsequencing and cDNA have independently shown the presence of a 16 amino acid N-terminal extension that is unique to the 120 kDa protein. Analysis of the myosin I beta gene has uncovered the presence of 2 new exons and demonstrated the basis, at the nucleotide level, for the differences in the nuclear and cytoplasmic forms of myosin I beta. Interestingly, a FLAG epitope attached to the cDNA for nuclear myosin I localizes in the nucleus but the FLAG epitope is found only in the cytoplasm when attached to the cytoplasmic myosin I. We have also produced antibodies to the 16 amino acid peptide and shown that they stain the nucleus. Taken together, these data provide strong support for the presence of myosin I in the nucleus and suggest that the 16 amino acid extension unique to nuclear myosin I is responsible for the nuclear localization of this protein. We now propose 3 Specific Aims that have the long range goal of defining the physiological functions of nuclear myosin I. They will, in order, address the following questions: (a) How do we get a nuclear myosin I beta protein? (b) How does this specific myosin I beta isoform get into the nucleus? (c) What does it do in the nucleus? Specific Aim 1 is a continuation of our ongoing characterization of the myosin I beta gene. Our analysis of this gene has suggested that the nuclear and cytoplasmic myosin I beta isoforms result from multiple mRNA species derived from the single gene on chromosome 11. Specific Aim 1 will test this hypothesis. The goal of Specific Aim 2 is to uncover the mechanisms that direct nuclear myosin I, but not other isoforms of myosin 1beta, to the nucleus. It will test the hypothesis that the 16 amino acid extension targets myosin I to the nucleus. Specific Aim 3 will investigate the physiological functions of myosin I in the nucleus by testing the hypothesis that nuclear myosin I has a structural role and/or the functioning of actively transcribing genes. Addressing these Specific Aims will help us to reach our ultimate goal of understanding the physiological functions of nuclear myosin I. This proposal will also develop a number of tools (eg: plasmids, monoclonal and anti-peptide antibodies, proteins expressed in baculovirus, knock-out cells) that will be instrumental in realizing our long range goal.